Represses Cyclin D1 Expression Research Articles

MCPIP1-mediated NFIC alternative splicing inhibits proliferation of triple-negative breast cancer via cyclin D1-Rb-E2F1 axis

Triple-negative breast cancer (TNBC) is the most aggressive subtype with the worst prognosis and the highest metastatic and recurrence potential, which represents 15–20% of all breast cancers in Chinese females, and the 5-year overall survival rate is about 80% in Chinese women. Recently, emerging evidence suggested that aberrant alternative splicing (AS) plays a crucial role in tumorigenesis and progression. AS is generally controlled by AS-associated RNA binding proteins (RBPs). Monocyte chemotactic protein induced protein 1 (MCPIP1), a zinc finger RBP, functions as a tumor suppressor in many cancers. Here, we showed that MCPIP1 was downregulated in 80 TNBC tissues and five TNBC cell lines compared to adjacent paracancerous tissues and one human immortalized breast epithelial cell line, while its high expression levels were associated with increased overall survival in TNBC patients. We demonstrated that MCPIP1 overexpression dramatically suppressed cell cycle progression and proliferation of TNBC cells in vitro and repressed tumor growth in vivo. Mechanistically, MCPIP1 was first demonstrated to act as a splicing factor to regulate AS in TNBC cells. Furthermore, we demonstrated that MCPIP1 modulated NFIC AS to promote CTF5 synthesis, which acted as a negative regulator in TNBC cells. Subsequently, we showed that CTF5 participated in MCPIP1-mediated antiproliferative effect by transcriptionally repressing cyclin D1 expression, as well as downregulating its downstream signaling targets p-Rb and E2F1. Conclusively, our findings provided novel insights into the anti-oncogenic mechanism of MCPIP1, suggesting that MCPIP1 could serve as an alternative treatment target in TNBC.

Circular RNA hsa_circ_0007059 restrains proliferation and epithelial-mesenchymal transition in lung cancer cells via inhibiting microRNA-378

As newly discovered non-coding RNA (ncRNA), circular RNA (circRNA) has become a research hotspot in manifold cancers. But, the influences of hsa_circ_0007059 in lung cancer remain obscure. Expression of hsa_circ_0007059 in lung cancer tissues was firstly determined through RT-qPCR. After overexpressing hsa_circ_0007059, cell viability, apoptosis, p53/CyclinD1, Bax and Pro/Cleaved-Caspase-3 and EMT-correlative factors (E-cadherin, Vimetin, Twist1 and Zeb1) were tested in A549 and H1975 cells. MiR-378 expression in lung cancer tissues and cells was evaluated after miR-378 mimic transfection. Wnt/β-catenin and ERK1/2 pathways were finally evaluated in A549 and H1975 cells. Inhibition of hsa_circ_0007059 was discovered in lung cancer tissues. Overexpressed hsa_circ_0007059 evidently restrained cell proliferation, elevated p53 and repressed CyclinD1 expression, meanwhile triggered apoptosis and enhanced Bax and Cleaved-Caspase-3 expression. Increased hsa_circ_0007059 abated EMT via enhancement of E-cadherin and inhibition of Vimentin, Twist and Zeb1 in A549 and H1975 cells. MiR-378 was up-regulated in lung cancer tissues, declined by hsa_circ_0007059 overexpression in A549 and H1975 cells. Overexpressed hsa_circ_0007059 hindered Wnt/β-catenin and ERK1/2 pathways via suppressing miR-378 in A549 and H1975 cells. The investigations manifested that hsa_circ_0007059 abated cell proliferation and EMT process in lung cancer cells via inactivation of Wnt/β-catenin and ERK1/2 pathways via suppressing miR-378.

The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo.

Oral squamous cell carcinoma (OSCC) is a common malignancy with a growing worldwide incidence and prevalence. The N-myc downstream regulated gene (NDRG) family of NDRG1, 2, 3, and mammary serine protease inhibitor (Maspin) gene are well-known modulators in the neoplasia process. Current research has considered iron chelators as new anti-cancer agents; however, the anticancer activities of iron chelators and their target genes in OSCC have not been well investigated. We showed that iron chelators (Dp44mT, desferrioxamine (DFO), and deferasirox) all significantly inhibit SAS cell growth. Flow cytometry further indicated that Dp44mT inhibition of SAS cells growth was partly due to induction of G1 cell cycle arrest. Iron chelators enhanced expressions of NDRG1 and NDRG3 while repressing cyclin D1 expression in OSCC cells. The in vivo antitumor effect on OSCC and safety of Dp44mT were further confirmed through a xenograft animal model. The Dp44mT treatment also increased Maspin protein levels in SAS and OECM-1 cells. NDRG3 knockdown enhanced the growth of OECM-1 cells in vitro and in vivo. Our results indicated that NDRG3 is a tumor suppressor gene in OSCC cells, and Dp44mT could be a promising therapeutic agent for OSCC treatment.

Aberrant expression of cyclin D1 in cancer.

Cyclin D1 binds and activates cyclin-dependent kinases 4/6 (Cdk4/6) to phosphorylate the retinoblastoma (RB) family proteins, relieving E2F/DPs from the negative restraint of RB proteins and histone deacetylases. The cyclin D-Cdk4/6 complexes activate cyclin E/Cdk2 through titration of the Cdk inhibitors p21Cip1/p27Kip1. Cyclin E/Cdk2 further phosphorylates RBs, thereby activating E2F/DPs, and cells enter the S phase of the cell cycle. Cyclin D-Cdk4/6 also phosphorylates MEP50 subunit of the protein arginine methyltransferase 5 (PRMT5), which cooperates with cyclin D1 to drive lymphomagenesis in vivo. Activated PRMPT5 causes arginine methylation of p53 to suppress expression of pro-apoptotic and anti-proliferative target genes, explaining the molecular mechanism for tumorigenesis. Cyclin D1 physically interacts with transcription factors such as estrogen receptor, androgen receptor, and Myb family proteins to regulate gene expression in Cdk-independent fashion. Dmp1 is a Myb-like protein that quenches the oncogenic signals from activated Ras or HER2 by inducing Arf/p53-dependent cell cycle arrest. Cyclin D1 binds to Dmp1α to activate both Arf and Ink4a promoters to induce cell cycle arrest or apoptosis in non-transformed cells to prevent them from neoplastic transformation. Dmp1-deficiency significantly accelerates mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Cyclin D1 interferes with ligand activation of PPARγ involved in cellular differentiation; it also physically interacts with histone deacetylases (HDACs) and p300 to repress gene expression. It has also been shown that cyclin D1 accelerates tumorigenesis through transcriptional activation of miR-17/20 and Dicer1 which, in turn, represses cyclin D1 expression. Identification of cyclin D1-binding proteins/promoters will be essential for further clarification of its biological activities.

Abstract ED05-03: Rexinoid nuclear receptors and cancer prevention

Abstract This session will present on the use of retinoid X receptor (RXR) agonists (rexinoids) for cancer prevention. Several areas will be highlighted. Using lung cancer as a model, both preclinical and clinical evidence for use of RXR-selective ligands in cancer prevention will be discussed. Emphasis will be placed on the pharmacology of newer rexinoids. Lung cancer is the most common cause of cancer death for men and women. Innovative ways to combat lung cancer are needed. A previously unrecognized antineoplastic pathway that triggers proteasomal degradation of different cyclins was found. Both retinoic acid receptor (RAR) and RXR agonists (among other agents) cause degradation of cyclins. This confers check point arrest and repair of carcinogenic damage of DNA in bronchial epithelial cells. Mechanisms responsible for this induced proteasomal degradation will be presented. The critical receptor that confers cyclin destabilization is RARβ. Yet, silencing of RARβ and of the novel isoform RARβ1' likely accounts for clinical resistance to classical retinoids in lung carcinogenesis. It was explored whether RXR/RAR heterodimer complex activation with a rexinoid triggers G1 cyclin destabilization. This was found and this result implied that the same pathway would be engaged in vivo. To establish if this occurred in the lung, transgenic mice were independently engineered with human surfactant C-driven wild-type cyclin E or a proteasome-degradation resistant cyclin E species. This was done because human pre-malignant and malignant lung lesions deregulate cyclin expression. Intriguingly, these mice recapitulated frequent features of lung carcinogenesis found in patients. These changes were enhanced by the expression of degradation-resistant cyclin E species. This finding was built upon by showing that a rexinoid can chemoprevent lung cancers in vinyl carbamate-induced lung tumors within strain A/J mice. The epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) erlotinib also repressed cyclin D1 expression, but through different mechanisms. The rexinoid bexarotene was combined with erlotinib. Effects on transgenic lung cancer cell growth and cyclin D1 expression were studied when KRAS or KRAS/p53 mutations were present or not. Over the past decade, this work was confirmed by extending analyses into the cancer clinic through a series of five clinical trials that moved this work through successive phase 0, phase I, and phase II trials. In window of opportunity trials, pharmacodynamic responses (cyclin repression and induced necrosis and inflammatory responses) were seen when intratumoral drug levels were comparable to those detected in vitro. Cyclin D1 levels were measured in pre-treatment versus post-treatment tumor biopsies. A phase II trial in heavily pre-treated stage IV non-small cell lung cancer (NSCLC) cases was performed. EGFR expression and KRAS mutations were examined in these cases. Findings revealed substantial cyclin repression and reduction of lung cancer growth by combined treatment with a rexinoid and EGFR-TKI. Cyclins were repressed while necrosis and inflammatory responses were induced in the window of opportunity trial independent of KRAS or activating EGFR mutations. This refractory NSCLC trial had 3 major clinical responses (2 had KRAS or EGFR mutations) with prolonged survival, as will be discussed. Median survival was 22 weeks (16 weeks for controls). Hypertriglyceridemia or rash significantly increased median overall survival to 24 weeks. This regimen revealed anti-tumor activity independent of the presence of EGFR or KRAS mutations in NSCLCs. Notably, these findings were independently replicated in the lung cancer BATTLE trial. An analogous approach was used to uncover a distinct rexinoid-induced protein destabilization pathway that involves the deubiquitinase USP18/UBP43, as will be discussed. Taken together, these findings reveal that rexinoid-induced cyclin destabilization is a previously unrecognized antineoplastic pathway. Thus, additional rexinoid trials for cancer therapy and prevention are warranted. Citation Format: Ethan Dmitrovsky. Rexinoid nuclear receptors and cancer prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr ED05-03.

Role of NF-κB–p53 crosstalk in ultraviolet A-induced cell death and G1 arrest in human dermal fibroblasts

Photoaging is the premature aging of the skin caused by repeated exposure to sunlight and is characterized by a depletion of the dermal extracellular matrix. This depletion is due to the loss of fibroblast cells and their multiple functions. UVA was revealed as a major inducer of photoaging in various clinical studies. As UVA photons have long wavelength spectra, UVA penetrates deeper into the dermis than UVB and UVC, leading to the induction of cell death, the destruction of the dermal extracellular matrix through the induction of matrix metalloproteinase expression, and the repression of collagen expression. However, the exact effects of UVA on the skin remain a matter of debate. Here, we assess cell cycle stage to demonstrate that NF-κB-p53 crosstalk induces apoptosis and growth arrest in UVA-irradiated human dermal fibroblasts. In addition, UVA irradiation led to an increase of NF-κB-HDAC1 complexes, which in turn repressed cyclin D1 expression in UVA-irradiated human dermal fibroblasts. We provide direct evidence that UVA irradiation induces changes in the p53-dependent NF-κB complex that lead to growth arrest and apoptosis through the repression of cyclin D1. These studies uncovered that NF-κB-p53 crosstalk is a key regulator of UVA-dependent growth arrest and apoptosis.

A novel antioestrogen agent (3R,6R)-bassiatin inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression in 17β-oestradiol-treated MCF-7 cells

Bassiatin [(3R,6R)-bassiatin] was isolated from the endogenous fungus Fusarium oxysporum J8-1-2. We found that oestrogen-dependent cell growth was inhibited by bassiatin in MCF-7 ERα (oestrogen receptor α)-positive breast cancer cells. In addition, the mRNA and protein levels of ERα and the oestrogen-responsive gene cyclin D1 were down-regulated by bassiatin in the presence of 17β-oestradiol. Co-treatment of 17β-oestradiol and bassiatin increased phospho-cyclin D1 (Thr286), an indicator of cyclin D1 phosphorylation-dependent degradation. However, this effect was not obvious in the absence of 17β-oestradiol, suggesting that bassiatin may play a role in the metabolism of cyclin D1 by decreasing cyclin D1 protein expression in the presence of 17β-oestradiol. Cyclin D1, known as a key cell cycle regulator, regulates the transition of G1- and S-phase. Decreased cyclin D1 was found to be involved in bassiatin-induced MCF-7 cell cycle arrest. Collectively, our study showed that bassiatin induced cell cycle arrest and exerted an antioestrogen effect. It may prove to be a potential drug target for antioestrogen therapy in ERα-positive breast cancer.

Abstract 1655: The ubiquitin protease UBP43 is a target for lung cancer therapy and prevention

Abstract Lung cancer is the leading cause of cancer mortality for women and men in the United States. Given this, there is a need to find new targets to combat lung cancer. This study explores the E1-like ubiquitin-activating enzyme (UBE1L) that associates with the interferon-stimulated gene 15 (ISG15), which complexes with cyclin D1 and other specific proteins. The ubiquitin protease UBP43 removes ISG15 from conjugated proteins. The UBE1L-ISG15-UBP43 pathway was previously proposed to inhibit lung carcinogenesis by repressing cyclin D1 expression. Our prior work is extended here by reporting that UBP43 is enzymatically active in cells. Also, gain of UBP43 expression specifically stabilizes cyclin D1 while UBP43 knock-down destabilizes cyclin D1, but not cyclin E or other D-type cyclins. This occurs by regulating ISG15 complexes with cyclin D1. UBP43 effects on cyclin D1 were not antagonized by cycloheximide treatment. Whether the deconjugase UBP43 was a lung cancer target was independently addressed through engineered gain and loss of UBP43 expression in lung cancer cells. UBP43 knock-down triggered apoptosis in these cells. In contrast, UBP43 over-expression promoted lung cancer cell growth by inhibiting apoptosis. That cyclin D1 plays a key role in conferring these effects was shown by engineered loss of UBP43 along with forced cyclin D1 expression. Cyclin D1 antagonized effects of loss of UBP43. Engineered UBP43 knock-down in lung cancer cells significantly increased apoptosis (P < 0.05), reduced growth (P < 0.05) and inhibited lung cancer formation (P < 0.05) in FVB mice injected via tail veins with syngeneic lung cancer cells. In marked contrast, forced UBP43 over-expression in lung cancer cells antagonized the effects of interferon, cisplatin and all-trans-retinoic acid, indicating that UBP43 can regulate response to anti-neoplastic agents. To ascertain the clinical impact of this pathway, a paired normal-malignant human lung tissue array from 74 cases was examined for immunohistochemical expression profiles of UBP43 and cyclin D1 proteins. Notably, UBP43 was significantly increased in the malignant as compared to the adjacent normal lung tissues (P = 0.04 for adenocarcinoma and P = 0.02 for squamous cell carcinoma). Intriguingly, a direct relationship was found between UBP43 and cyclin D1, validating clinical relevance. Thus, UBP43 knock-down appears to exert its anti-neoplastic effects by destabilizing cyclin D1. Taken together, these findings establish that the deconjugase UBP43 is a tractable target for lung cancer therapy and prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2011-1655

Abstract 342: Murine transgenic lung cancer models guide a proof of principle histone deacetylase inhibitor trial in lung cancer patients

Abstract Lung cancer is the most common cause of cancer mortality in the United States. New treatments for lung cancer are therefore needed. We previously engineered murine transgenic cyclin E models that recapitulated frequent features of lung cancers in patients. These included chromosome instability, hedgehog pathway and cyclin D1 activation as well as single or multiple pre-malignant and malignant (adenocarcinoma) lung lesions. Metastases were also observed. Cell lines (ED-1 and ED-2) were derived from these murine lung cancers. After tail vein injection of ED-1 or ED-2 cells into syngeneic FVB mice, neoplastic lung lesions form within 1-2 weeks. These models are useful tools to identify promising anti-neoplastic agents. Among a panel of drugs that caused growth inhibition of lung cancer cells and repression of cyclin D1 protein, the histone deacetylase inhibitor (Vorinostat) was found as most potent. Vorinostat treatment caused dose- (1-5μM) and time-dependent (2 and 4 days) growth inhibition and apoptosis induction in murine lung cancer cells. Effects were reversed by washouts of Vorinostat. Similar reversible Vorinostat effects on growth inhibition and apoptosis were observed in human lung cancer cell lines (HOP62, H522 and H23). Vorinostat treatments repressed cyclin D1 and cyclin E proteins, but increased p27 expression in ED-1 cells. Notably, Vorinostat treatments of cyclin E transgenic mice also substantially repressed cyclin D1 expression in pre-malignant and malignant lung tissues. Vorinostat treatment also significantly reduced lung tumor formation (p = 0.047) in FVB mice harboring transplanted syngeneic murine lung cancer cells. To translate studies into the clinic, an Institutional Review Board-approved proof of principle Vorinostat trial was launched for early stage non-small cell lung cancer cases undergoing surgical resection. Patients receive Vorinostat treatments pre-operatively. Post- versus pre-treatment biopsies will be scored for changes in immunohistochemical profiles of cyclin D1, cyclin E, p27, and ki-67. Trial accrual ends January 2011. Results will then be analyzed with findings reported at this meeting. Taken together, Vorinostat treatments repressed lung cancer cell growth by augmenting apoptosis and reducing cyclin D1 expression. Substantial anti-neoplastic effects of Vorinostat were seen in murine transgenic and transplantable lung cancer models. These studies underscore the value of clinically-relevant murine lung cancer models for guiding clinical trials in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2011-342

Moesin–ezrin–radixin-like protein (merlin) mediates protein interacting with the carboxyl terminus-1 (PICT-1)-induced growth inhibition of glioblastoma cells in the nucleus

Moesin–ezrin–radixin-like protein (merlin) has long been considered a unique tumour suppressor that inhibits mitogenic signalling only at the membrane–cytoskeleton interface. However, the nucleocytoplasmic shuttling of merlin in a cell cycle-dependent manner has recently been observed, indicating that merlin may also exert its tumour-suppressive activity by interacting with specific nuclear protein partners. We have identified protein interacting with carboxyl terminus 1 (PICT-1) as a novel merlin-binding partner. Although the detailed mechanisms are not fully understood, several lines of evidence have previously implicated PICT-1 as a candidate tumour suppressor, including its phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-dependent growth-suppression and cell-killing activities. We show here that PICT-1 is localised to the nucleolus, and Ser518-dephosphorylated merlin (the growth-inhibitory form of merlin) can interact with PICT-1 in the nucleolus. Ectopic expression of PICT-1, both in PTEN-positive HeLa cells and in PTEN-deficient U251 cells, effectively represses cyclin D1 expression, arrests the cell cycle at G0/G1, and promotes cell apoptosis. PICT-1 (1–356), a carboxyl-terminus truncated mutant that has lost the ability to bind merlin, has a markedly reduced inhibitory effect on the cell cycle and proliferation. Knockdown of merlin expression by siRNA attenuates the inhibitory effects induced by PICT-1 over-expression. We propose that merlin mediates PICT-1-induced growth inhibition by translocating to the nucleolus and binding PICT-1.

Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

A Jumonji (Jarid2) Protein Complex Represses cyclin D1 Expression by Methylation of Histone H3-K9

Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

A role for candidate tumor-suppressor gene TCEAL7 in the regulation of c-Myc activity, cyclin D1 levels and cellular transformation

The pathophysiological mechanisms that drive the development and progression of epithelial ovarian cancer remain obscure. Recently, we identified TCEAL7 as a transcriptional regulatory protein often downregulated in epithelial ovarian cancer. However, the biological significance of such downregulation in cancer is not currently known. Here, we show that TCEAL7 is downregulated frequently in many human cancers and that in immortalized human ovarian epithelial cells this event promotes anchorage-independent cell growth. Mechanistic investigations revealed that TCEAL7 associates with cyclin D1 promoter containing Myc E-box sequence and transcriptionally represses cyclin D1 expression. Moreover, downregulation of TCEAL7 promotes DNA-binding activity of Myc-Max, and upregulates the promoter activity of c-Myc-target gene, ornithine decarboxylase (ODC), whereas enhanced expression of TCEAL7 inhibits Myc-induced promoter activity of ODC. Our findings suggest that TCEAL7 may restrict ovarian epithelial cell transformation by limiting Myc activity. These results also suggest a potential, alternative mechanism by which c-Myc activity may be deregulated in cancer by the downregulation of TCEAL7.

Combined effects of retinoic acid and histone deacetylase inhibitors on human neuroblastoma SH-SY5Y cells

All-trans retinoic acid (RA) causes differentiation of neuroblastoma cells, and retinoids have been used in clinical trials in children with advanced neuroblastoma. Combination of RA with histone deacetylase inhibitors (HDACi) could result in improved antitumorigenic activity. We have examined the effect of the HDACi trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA), alone and in combination with RA in human neuroblastoma SH-SY5Y cells. At concentrations that cause sustained increase of histone H3 acetylation, HDACi produced extensive apoptotic cell death as shown by flow cytometry analysis and induction of poly(ADP-ribose) polymerase proteolysis. HDACi inhibited SH-SY5Y cell growth at a much larger extent than RA. This compound did not cause apoptosis and did not further increase HDACi-mediated cell death. In contrast, both types of drugs cooperated to inhibit cell growth, although synergistic effects were not found. In surviving cells, HDACi repressed cyclin D1 expression and increased the cyclin kinase inhibitors (CKI) p21(Waf1/Cip1) and p27(Kip1). Cyclin D1 was not affected by RA, but this retinoid also increased CKI levels. Induction of p21(Waf1/Cip1) and p27(Kip1) by HDACi was further enhanced in the presence of RA. This effect seems to be at least partially due to transcriptional stimulation of CKI gene expression because both types of drugs cooperated to increase CKI mRNA levels and to activate the CKI promoters in transient transfection assays. These results show the strong antitumorigenic effects of HDACi in neuroblastoma cells and reinforce the idea that combination therapy could be useful to inhibit tumor growth.

Bexarotene and Erlotinib for Aerodigestive Tract Cancer

The epidermal growth factor receptor (EGFR) and cyclin D1 are overexpressed in lung carcinogenesis. The rexinoid, bexarotene, represses cyclin D1 and EGFR expression in vitro. It was hypothesized that combining bexarotene with the EGFR inhibitor, erlotinib, would augment clinical activity. In vitro studies and a phase I clinical trial were performed. Twenty-four patients with advanced aerodigestive tract cancers were enrolled; 79% had non-small-cell lung cancer (NSCLC). The primary objective was to determine the maximum-tolerated dose. Clinical activity was a secondary objective. Combining erlotinib with bexarotene enhanced growth suppression in vitro compared with each single-agent treatment. This cooperatively repressed cyclin D1 expression. Clinically, the most frequent toxicities were mild hypertriglyceridemia and skin rash. Two serious treatment-related adverse events occurred (creatine phosphokinase elevation attributed to antilipid therapy and a case of generalized pain). Five objective responses (four partial and one minor) were observed in NSCLC patients. Responses were observed in males and smokers. EGFR sequence analyses did not reveal activating mutations in tumors from assessable responding patients. Median time to progression was 2.0 months; overall survival time was 14.1 months; and 1-year survival rate was 73.8%. The recommended phase II doses are erlotinib 150 mg/d and bexarotene 400 mg/m2/d orally. These agents can be administered in combination at the recommended single-agent doses without added toxicity. Overall survival and clinical features of responding patients differ from prior reports of single-agent erlotinib treatment. These findings are encouraging and warrant further investigation of this regimen.

The NF2 Tumor Suppressor Gene Product, Merlin, Inhibits Cell Proliferation and Cell Cycle Progression by Repressing Cyclin D1 Expression

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.

Microarray Analyses Uncover UBE1L as a Candidate Target Gene for Lung Cancer Chemoprevention

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.

The inhibitor of cyclin-dependent kinase 4a/alternative reading frame (INK4a/ARF) locus encoded proteins p16INK4a and p19ARF repress cyclin D1 transcription through distinct cis elements.

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF) (murine p19(ARF)). Invariant inactivation of either the p16(INK4a)-cyclin D/CDK-pRb pathway and/or p53-p14(ARF) pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16(INK4a)/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1-/- mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16(INK4a) expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16(INK4a) was observed in tissues of mice mutant for the Ink4a/Arf locus. p16(INK4a) and p19(ARF) inhibited DNA synthesis in MCF7 cells. p16(INK4a) repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16(INK4a) occurred independently of the p16(INK4a)-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19(ARF) repressed cyclin D1 through a novel distal cis-element at -1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

Modifiers of the jumonji mutation downregulate cyclin D1 expression and cardiac cell proliferation

Cell proliferation is an important factor in various developmental processes in tissue morphogenesis, and is strictly regulated spatiotemporally. jumonji ( jmj) deficient mice with a C3H/He background show hyperproliferation of cardiac myocytes and die probably of the phenotype around embryonic day 11.5. Analyses of the abnormalities revealed that repression of cyclin D1 expression by jmj is necessary for downregulation of cardiac myocyte proliferation. On the other hand, jmj mutant mice with a BALB/c background die around E14.5, suggesting that genetic background modifies hyperproliferation in the heart and timing of lethality. Here, we demonstrated that the hyperproliferation was not observed, and that cell proliferation and expression of cyclin D1 were downregulated properly in the cardiac ventricles of jmj mutant mice with a BALB/c background. These results suggest the modifier(s) of the jmj mutation can downregulate cardiac cell proliferation by repressing cyclin D1 expression in the same way as jmj.

Jumonji Downregulates Cardiac Cell Proliferation by Repressing cyclin D1 Expression

Spatiotemporal regulation of cell proliferation is necessary for normal tissue development. The molecular mechanisms, especially the signaling pathways controlling the cell cycle machinery, remain largely unknown. Here, we demonstrate a negative relationship between the spatiotemporal patterns of jumonji ( jmj) expression and cardiac myocyte proliferation. cyclin D1 expression and cell proliferation are enhanced in the cardiac myocytes of jmj-deficient mutant embryos. In contrast, jmj overexpression represses cyclin D1 expression in cardiac cells, and Jmj protein binds to cyclin D1 promoter in vivo and represses its transcriptional activity. cyclin D1 overexpression causes hyperproliferation in the cardiac myocytes, but the absence of cyclin D1 in jmj mutant embryos rescues the hyperproliferation. Therefore, Jmj might control cardiac myocyte proliferation and consequently cardiac morphogenesis by repressing cyclin D1 expression.